Engineering ‘designer’ glycomodules for boosting recombinant protein secretion in tobacco hairy root culture and studying hydroxyproline‐O‐glycosylation process in plants

Abstract

Summary The key technical bottleneck for exploiting plant hairy root cultures as a robust bioproduction platform for therapeutic proteins has been low protein productivity, particularly low secreted protein yields. To address this, we engineered novel hydroxyproline (Hyp)‐ O ‐glycosylated peptides (Hyp GP s) into tobacco hairy roots to boost the extracellular secretion of fused proteins and to elucidate Hyp‐ O ‐glycosylation process of plant cell wall Hyp‐rich glycoproteins. Hyp GP s representing two major types of cell wall glycoproteins were examined: an extensin module consisting of 18 tandem repeats of ‘Ser‐Hyp‐Hyp‐Hyp‐Hyp’ motif or ( SP 4) 18 and an arabinogalactan protein module consisting of 32 tandem repeats of ‘Ser‐Hyp’ motif or ( SP ) 32 . Each module was expressed in tobacco hairy roots as a fusion to the enhanced green fluorescence protein ( EGFP ). Hairy root cultures engineered with a Hyp GP module secreted up to 56‐fold greater levels of EGFP , compared with an EGFP control lacking any Hyp GP module, supporting the function of Hyp GP modules as a molecular carrier in promoting efficient transport of fused proteins into the culture media. The engineered ( SP 4) 18 and ( SP ) 32 modules underwent Hyp‐ O ‐glycosylation with arabino‐oligosaccharides and arabinogalactan polysaccharides, respectively, which were essential in facilitating secretion of the fused EGFP protein. Distinct non‐Hyp‐ O ‐glycosylated ( SP 4) 18 ‐ EGFP and ( SP ) 32 ‐ EGFP intermediates were consistently accumulated within the root tissues, indicating a rate‐limiting trafficking and/or glycosylation of the engineered Hyp GP modules. An updated model depicting the intracellular trafficking, Hyp‐ O ‐glycosylation and extracellular secretion of extensin‐styled ( SP 4) 18 module and AGP ‐styled ( SP ) 32 module is proposed.

Fusion of a Novel Native Signal Peptide Enhanced the Secretion and Solubility of Bioactive Human Interferon Gamma Glycoproteins in Nicotiana benthamiana Using the Bamboo Mosaic Virus-Based Expression System

“…These results are in agreement with the previous studies for the production of high amount of secreted recombinant proteins in tobacco BY2 suspension cells (Xu et al, 2007(Xu et al, , 2010Zhang et al, 2019a), tobacco hairy root cultures (Zhang et al, 2019b) and green microalga Chlamydomonas reinhardtii (Ramos-Martinez et al, 2017). HypGP modules have been shown to be molecular carriers to boost the transport of fused proteins along secretion pathway into extracellular space (Zhang et al, 2019b). Interestingly, in our study, both the yield and solubility of SS Ext mIFNγ(SP) 10 were increased significantly, up to 6 and 2.7 folds, respectively, higher than those from the N. benthamiana leaf tissues expressing the control mIFNγ (Table 1 and Figure 2C).…”

A secreted protein of 15 kDa plays an important role in Phytophthora palmivora development and pathogenicity

“…Their results suggest that a specific enzymatic cleavage of sugar or amino acid residues may be responsible for double bands 41 . Zhang et al 42 expressed the cell wall glycoprotein (SP) 32 fused with green fluorescence protein in the leaves and hairy roots of tobacco (N. tabacum). They observed two bands of 40 and 42 kDa.…”

“…They observed two bands of 40 and 42 kDa. The 42 kDa band disappeared when the expressed product was subjected to β-elimination treatment known to remove sugars O-linked to Ser/ Thr 42 . When N-glycosylated cholera toxin B subunit (gCTB) was transiently expressed in N. benthamiana leaves, Western blot analysis of the expressed protein without glycosidase treatment showed two distinct bands 43,44 .…”

High Efficient Expression and Purification of Human Epidermal Growth Factor in Arachis Hypogaea L.

“…Hairy root is a good host for mass production of recombinant protein due to its abundant quality and extensive lateral branches [19]. Hairy root production system has received considerable interest in recent years [20,21]. As promising systems for protein production, hairy root cultures have been used to obtain vaccine, antibody, and multiple clinical proteins [22,23].…”